MSACL 2025 Abstract

Development and ‘Fit-for-Purpose’ Analytical Validation of a High-Sensitivity pTau217 Mass Spec Assay in Human Plasma

Erpan Ahat, Erin Wray, Joel Cook, Shamili Sammohi, Sarika Chauhan, Gary Heady, Michael Hodsdon
Clinical Diagnostics Laboratory, Eli Lilly and Company, Indianapolis, Indiana

Erpan Ahat, PhD (Presenter) Eli Lilly and Company

Presenter Bio: Erpan obtained his PhD from the University of Michigan in December 2021 majoring in Molecular and cellular biology studying Golgi mediated protein post-translational modification and secretion. In his current role in the Clinical Diagnostics Lab (CDL) at Lilly, Erpan develops mass spec-based biomarker assays to support the mission of CDL which is to deliver diagnostic solutions to enhance disease understanding and guide optimal patient treatment.

Relevant Financial Disclosures (within past 24 months, reported on May 27, 2025) Other Potential Conflicts Eli Lilly & Company / Employee Eli Lilly & Company / minor shareholder Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

INTRODUCTION
Tau exists in various physiological and pathological proteoforms that regulate Alzheimer's disease (AD) progression. These proteoforms, when released from neurons into the periphery, serve as potential biomarkers. Phosphorylated tau species, particularly at positions T181, T217, and T231, have emerged as crucial biomarkers for AD in cerebrospinal fluid (CSF) and plasma-based assays, with P217 tau demonstrating superior diagnostic utility.

OBJECTIVE
To develop and validate a mass spectrometry-based p-tau217 assay featuring enhanced specificity and multiplexing capabilities for comprehensive AD pathology staging.

METHODS
The IP-MS p-tau217 assay employs a streamlined workflow incorporating:
• Immunoprecipitation with a p-tau217 specific antibody on KingFisher Apex platform
• Sample processing through SPE-based desalting and clean-up using OTTO
• Enzymatic digestion with trypsin
• Quality monitoring through dual heavy standard controls:
• Winged peptide standard introduced before IP
• AQUA heavy standard added post-digestion for quality monitoring

RESULTS
Analytical Performance
• Demonstrated robust precision with total CV of ~10% across 130 measurements over 10 days
• Excellent accuracy with spike recovery of 92.4-97.9%
• Strong parallelism ranging from 89.1-98.3%
Analytical Sensitivity
• LOB: 0.015 fmol/mL
• LOD: 0.02 fmol/mL
• LLOQ: 0.03 fmol/mL
• AMR: 0.03-2.29 fmol/mL
Sample Stability
• Up to 72-hour stability at room temperature
• Up to 48-hour stability at 4°C
• Integrity maintained through five freeze-thaw cycles
• Minimal impact from preanalytical variables (hemolysis, lipemia, non-phospho tau)
Clinical Utility
• Strong correlation with validated internal ELISA-based assays (R²>0.9)
• Potentially enhanced performance in cognitively normal population through superior specificity
• Unique multiplexing capability for comprehensive pathology assessment

CONCLUSION
This validated IP-MS p-tau217 assay represents a significant advancement in AD biomarker quantification, combining enhanced specificity through high-resolution mass spectrometry with comprehensive multiplexing capabilities. The method demonstrates exceptional analytical performance, accuracy, and improved specificity compared to conventional immunoassays.